Engineered botulinum neurotoxin

ABSTRACT

Disclosed herein are  botulinum  neurotoxin (BoNT) polypeptides with a modified receptor binding domain of Clostridial  botulinum  serotype B (B-H c ), comprising one or more substitution mutations corresponding to substitution mutations in serotype B, strain 1, V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; SI 20 IV; or combinations thereof. Specific combination mutations include E1 191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S 1199Y, or E 1191 Q and S 1199F. Other substitution mutations are also disclosed. Isolated modified receptor binding domains, chimeric molecules, pharmaceutical compositions, and methods of using the same are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/653,214, filed May 30, 2012, the contentsof which is incorporated herein by reference in its entirety.

GOVERNMENTAL SUPPORT

This invention was made with Government support under NCRR RR000168awarded by the National Institute of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutics forneuromuscular disorders.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins are a family of bacterial toxins, including sevenmajor serotypes (BoNT/A-G) ¹. These toxins act by blockingneurotransmitter release from neurons, thus paralyzing animals andhumans. In recent years, BoNTs have been widely used to treat a growinglist of medical conditions: local injections of minute amount of toxinscan attenuate neuronal activity in targeted regions, which can bebeneficial in many medical conditions as well as for cosmetic purposes²⁻⁴.

BoNT/A and BoNT/B are the only two BoNTs that are currently FDA-approvedfor use in humans ²⁻⁴. These are toxins purified from bacteria withoutany sequence modifications (defined as wild type, WT). As theapplication of BoNTs grows, limitations and adverse effects have beenreported. The major limitation is the generation of neutralizingantibodies in patients, which renders future treatment ineffective ⁵.Termination of BoNT usage often leaves patients with no other effectiveways to treat/relieve their disorders. The possibility of antibodyresponses is directly related to both toxin doses and the frequency ofinjection ⁵. Therefore, this limitation mainly occurs in treating musclespasms, which involves relatively high doses of toxins. Consistently,antibody responses have not been observed in cosmetic applications,which use extremely low toxin doses ⁵.

The major adverse effects are also often associated with treating musclespasms, but not cosmetic applications. This is because the adverseeffects are largely due to diffusion of toxins to other regions of thebody and the possibility of toxin diffusion is directly related toinjected doses. The adverse effects ranges from transient non-seriousevents such as ptosis and diplopia to life-threatening events even death^(6,7). In a petition letter filed in 2008 by Dr. Sidney Wolfe to FDA, atotal of 180 serious adverse events, including 16 deaths have beendocumented. As a result, FDA now requires the “Black box warning” on allBoNT products, highlighting the risk of the spread of toxins, followingsimilar warnings issued by the European Union.

Because both the generation of neutralizing antibodies and toxindiffusion are directly related to injected doses, lowering toxin doses(while maintaining the same levels of toxin activity) is highly desired,which means the efficacy of individual toxin molecules has to beenhanced. Such modified BoNTs with improved specificity for neurons willalso reduce any potential off-target effects due to non-specific entryinto other cell types.

BoNTs target and enter neurons by binding to their specific receptorsthrough their receptor binding domains, which are well-defined in theliterature (BoNT-H_(C), FIG. 1A, B) ¹. Receptor binding dictates theefficacy and specificity of BoNTs to recognize neurons. Improving thereceptor binding ability of BoNTs will enhance their efficacy andspecificity to target neurons. The receptors for most BoNTs have beenidentified (FIG. 1C). BoNT/B, D-C, and G share two homologous synapticvesicle proteins synaptotagmin I and II (Syt I/II) as their receptors⁸⁻¹³, while BoNT/A, E, D, and F use another synaptic vesicle protein SV2^(9,14-18). In addition to protein receptors, all BoNTs require lipidco-receptor gangliosides (FIG. 1D), which are abundant on neuronalsurfaces ¹⁹. Among the two Syt isoforms in rodents and likely in mostmammals, Syt II has ˜10-fold higher binding affinity for BoNT/B than SytI and is also the dominant isoform expressed in motor nerve terminals,which are the targeted neurons for BoNTs (FIG. 2A) ^(20,21.) Therefore,in rodents (on which most research has been conducted), Syt II isconsidered the major toxin receptor, while Syt I is a minor toxinreceptor at motor nerve terminals.

One may argue that BoNTs already have high specificity to neurons, is itpossible to further improve their binding to neurons? The answer is a“Yes” for humans, because it was recently discovered that the human SytII has greatly diminished binding and function as the receptor forBoNT/B due to a unique amino acid change from rodent (rat/mouse) Syt IIwithin the toxin binding site ^(13,22). This is a change fromphenylalanine (F) to leucine (L) at position 54 (mouse Syt II sequence)(FIG. 2B). Sequence alignments have revealed that phenylalanine at thisposition is highly conserved in both Syt I and Syt II acrossvertebrates, including platypus, fish, rodents, and monkeys ²³. Onlyhuman and chimpanzee Syt II contains leucine at this position. As aresult of this residue change, human and chimpanzee Syt II has greatlydiminished binding to BoNT/B, D-C, and G (FIG. 2C) and is significantlyless efficient in mediating the entry of BoNT/B (FIG. 2D), as comparedto mouse Syt II. Since human and chimpanzee Syt I still containsphenylalanine at the same position and can bind BoNT/B, D-C, and G (FIG.2E), the high affinity receptor for BoNT/B, D-C, and G in humans isrestricted to the minor receptor Syt I. These findings provide anexplanation for the clinical observations that a much higher dose ofBoNT/B than BoNT/A (which binds a different receptor) is needed toachieve the same levels of therapeutic effects in patients ^(24,25).Previously these observations were attributed to other reasons, such asthe percentage of active neurontoxin in the preparations used. Therecent observations of such binding differences of BoNT/B and human SytII versus Syt II of other species suggests that different residues ofBoNT/B may be involved in binding to human Syt II. As such, sequencemodification to BoNT/B that is expected to affect binding to rodentSytII may have unpredictable affects on BoNT/B binding to human Syt II.

SUMMARY

One aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide comprising a protease domain, a protease cleavage site, atranslocation domain, and a modified receptor binding domain ofClostridial botulinum serotype B (B-H_(c)), comprising one or moresubstitution mutations corresponding to substitution mutations inserotype B, strain 1, selected from the group consisting of V1118M;Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V;and combinations thereof. In one embodiment, the modified (B-H_(c))comprises two substitution mutations. In one embodiment, the twosubstitution mutations correspond to E1191M and S1199L, E1191M andS1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, orE1191Q and S1199F. In one embodiment, the two substitution mutationscorrespond to E1191M and S1199L. In one embodiment, the two substitutionmutations correspond to E1191M and S1199Y. In one embodiment, the twosubstitution mutations correspond to E1191M and S1199F. In oneembodiment, the two substitution mutations correspond to E1191Q andS1199L. In one embodiment, the two substitution mutations correspond toE1191Q and S1199Y. In one embodiment, the two substitution mutationscorrespond to E1191Q and S1199F.

Another aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide comprising a protease domain, a protease cleavage site, atranslocation domain, and a modified receptor binding domain ofClostridial botulinum serotype B (B-Hc), comprising a substitutionmutation at a position corresponding to S1199 or S1201 of serotype B,strain 1. In one embodiment, the substitution mutation produces enhancedbinding of the modified B-Hc to human SytII and/or reduced binding ofthe modified B-Hc to human Syt I as compared to an identical moleculelacking the substitution mutation. In one embodiment, the substitutionmutation produces enhanced binding of the modified B-Hc to human SytIIand/or increased binding of the modified B-Hc to human Syt I as comparedto an identical molecule lacking the substitution mutation. In oneembodiment, the substitution mutation is selected from the groupconsisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y and Vsubstituted for S. In one embodiment, the substitution mutation is anon-naturally occurring amino acid substituted for S. In one embodiment,the modified B-Hc is of strain 1. In one embodiment, the proteasedomain, translocation domain, and protease cleavage site are fromserotype selected from the group consisting of A, B, C, D, E, F, G, andcombinations thereof. In one embodiment, the protease domain,translocation domain, and protease cleavage site are from serotype B,strain 1. In one embodiment, the protease domain, translocation domain,and protease cleavage site are from serotype A, strain 1.

Another aspect of the invention relates to a polypeptide comprising amodified receptor binding domain of Clostridial botulinum serotype B(B-Hc) comprising one or more substitution mutations corresponding tosubstitution mutations in serotype B, strain 1, selected from the groupconsisting of V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y;S1199F; S1199L; S1201V; and combinations thereof. In one embodiment, themodified (B-Hc) comprises two substitution mutations. In one embodiment,the two substitution mutations correspond to E1191M and S1199L, E1191Mand S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, orE1191Q and S1199F. In one embodiment, the two substitution mutationscorrespond to E1191M and S1199L. In one embodiment, the two substitutionmutations correspond to E1191M and S1199Y. In one embodiment, the twosubstitution mutations correspond to E1191M and S1199F. In oneembodiment, the two substitution mutations correspond to E1191Q andS1199L. In one embodiment, the two substitution mutations correspond toE 1191Q and S1199Y. In one embodiment, the two substitution mutationscorrespond to E1191Q and S1199F.

Another aspect of the invention relates to a polypeptide comprising amodified receptor binding domain of Clostridial botulinum serotype B(B-Hc) comprising a substitution mutation at a position corresponding toS1199 or S1201 of serotype B, strain 1. In one embodiment, thesubstitution mutation produces enhanced binding of the modified B-Hc tohuman SytII and/or reduced binding of the modified B-Hc to human Syt Ias compared to an identical molecule lacking the substitution mutation.In one embodiment, the substitution mutation produces enhanced bindingof the modified B-Hc to human SytII and/or increased binding of themodified B-Hc to human Syt I as compared to an identical moleculelacking the substitution mutation. In one embodiment, the substitutionmutation is selected from the group consisting of A, R, N, D, C, Q, E,G, H, I, L, K, M, F, P, T, W, Y and V substituted for S. In oneembodiment, the substitution mutation is a non-naturally occurring aminoacid substituted for S. In one embodiment, the modified B-Hc is ofstrain 1.

Another aspect of the invention relates to a chimeric moleculecomprising a first portion that is a modified receptor binding domain ofClostridial botulinum serotype B (B-Hc) linked to a second portion,wherein the modified B-Hc comprises one or more substitution mutationscorresponding to substitution mutations in serotype B, strain 1,selected from the group consisting of V1118M; Y1183M; E1191M; E1191I;E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V and combinations thereof.In one embodiment, the modified B-Hc comprises two substitutionmutations. In one embodiment, the two substitution mutations correspondto E 1191M and S1199L, E 1191M and S1199Y, E 1191M and S1199F, E1191Qand S1199L, E1191Q and S1199Y, or E1191Q and S1199F. In one embodiment,the two substitution mutations correspond to E1191M and S1199L. In oneembodiment, the two substitution mutations correspond to E1191M andS1199Y. In one embodiment, the two substitution mutations correspond toE1191M and S1199F. In one embodiment, the two substitution mutationscorrespond to E1191Q and S1199L. In one embodiment, the two substitutionmutations correspond to E1191Q and S1199Y. In one embodiment, the twosubstitution mutations correspond to E1191Q and S1199F. In oneembodiment, the modified B-Hc comprises a modified receptor bindingdomain of Clostridial botulinum serotype B (B-Hc) comprising asubstitution mutation at a position corresponding to S1199 or S1201 ofserotype B, strain 1. In one embodiment, the substitution mutationproduces enhanced binding of the modified B-Hc to human SytII and/orreduced binding of the modified B-Hc to human Syt I as compared to anidentical molecule lacking the substitution mutation. In one embodiment,the substitution mutation produces enhanced binding of the modified B-Hcto human SytII and/or increased binding of the modified B-Hc to humanSyt I as compared to an identical molecule lacking the substitutionmutation. In one embodiment, the substitution mutation is selected fromthe group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, T,W, Y and V substituted for S. In one embodiment, the substitutionmutation is a non-naturally occurring amino acid substituted for S. Inone embodiment, the modified B-Hc is of strain 1. In one embodiment, thefirst portion and the second portion are linked covalently. In oneembodiment, the first portion and the second portion are linkednon-covalently. In one embodiment, the second portion is selected fromthe group consisting of a small molecule, a nucleic acid, a shortpolypeptide and a protein. In one embodiment, the second portion is abioactive molecule. In one embodiment, the second portion is atherapeutic polypeptide or non-polypeptide drug.

Another aspect of the invention relates to a nucleic acid comprising anucleotide sequence that encodes the polypeptide or chimeric moleculedescribed herein.

Another aspect of the invention relates to a nucleic acid vectorcomprising the nucleic acid described herein.

Another aspect of the invention relates to a cell comprising the nucleicacid vector described herein or the nucleic acid described herein.

Another aspect of the invention relates to a cell expressing thepolypeptide or chimeric molecule described herein.

Another aspect of the invention relates to a pharmaceutical compositioncomprising the botulinum neurotoxin (BoNT) polypeptide described herein,or the chimeric molecule described herein, or the nucleic acid vectordescribed herein, or the nucleic acid described herein. In oneembodiment, the pharmaceutical composition further comprises apharmaceutically acceptable excipient.

Another aspect of the invention relates to a kit comprising apharmaceutical composition described herein and directions fortherapeutic administration of the pharmaceutical composition.

Another aspect of the invention relates to a method to produce abotulinum neurotoxin (BoNT) polypeptide, the method comprising the stepsof culturing the host cell described herein under conditions whereinsaid BoNT polypeptide is produced. In one embodiment, the method furthercomprises recovering the BoNT polypeptide from the culture.

Another aspect of the invention relates to a method for treating acondition associated with unwanted neuronal activity comprisingadministering a therapeutically effective amount of the BoNT polypeptidedescribed herein to a subject to thereby contact one or more neuronsexhibiting unwanted neuronal activity, to thereby treat the condition.In one embodiment, the condition is selected from the group consistingof, spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia,oromandibular dysphonia, lingual dystonia, cervical dystonia, focal handdystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder,cerebral palsy, focal spasticity and other voice disorders, spasmodiccolitis, neurogenic bladder, anismus, limb spasticity, tics, tremors,bruxism, anal fissure, achalasia, dysphagia and other muscle tonedisorders and other disorders characterized by involuntary movements ofmuscle groups, lacrimation, hyperhydrosis, excessive salivation,excessive gastrointestinal secretions, secretory disorders, pain frommuscle spasms, headache pain, and dermatological or aesthetic/cosmeticconditions.

Another aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide described herein, the pharmaceutical composition ofdescribed herein, the chimeric molecule described herein, or thepolypeptide described herein, any one of which for use in a medicamentor medicine.

Another aspect of the invention relates to a botulinum neurotoxin (BoNT)polypeptide described herein, the pharmaceutical composition ofdescribed herein, the chimeric molecule described herein, or thepolypeptide described herein, any one of which for use in treating acondition associated with unwanted neuronal activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1D shows schematic models for how BoNTs target neurons (A),their overall protein structure (B), a list of identified receptors (C),and the structural model for BoNT/B binding to its receptors Syt andgangliosides (D). FIG. 1A) A schematic view of BoNT actions: BoNTsrecognize neurons by binding to their specific receptors (step 1), enterneurons via receptor-mediated endocytosis (step 2), the light chains ofBoNTs then translocate across endosomal membranes into the cytosol (step3), where these light chains act as proteases to cleave target hostproteins (step 4). Panel A is adapted from Amon, S. et al, JAMA,285:1059, 2001 ³³. FIG. 1B: BoNTs are composed of a light chain and aheavy chain, connected via a disulfide bond. The heavy chain can befurther divided into two domains: the translocation domain (H_(N)) andthe receptor binding domain (HC). These functional domains arewell-defined and switchable between different BoNTs ¹. This suggeststhat the modified BoNT/B-H_(C) can be used to replace BoNT/A-H_(C) togenerate chimeric toxins. FIG. 1C) A list of identified toxin receptors.FIG. 1D) A structural model showing binding of BoNT/B to its proteinreceptor, rodent Syt (I/II), as well as its lipid co-receptor,gangliosides, on the cell surface. D is adapted from Chai et al, Nature,444:1096, 2006³¹.

FIG. 2A-FIG. 2G show prior data adapted from published literaturesshowing (1) human Syt II is not an effective receptor for BoNT/B, D-C,and G; (2) residue changes in the receptor binding domain of BoNT/B cansignificantly change the binding affinity to Syt II and the potency oftoxins; (3) key residues within the receptor binding domain of BoNT/Bthat have been hypothesized to contribute to binding Syt II. FIG. 2A)The comparison between rodent Syt I and Syt II indicates that Syt II isthe major toxin receptor, while syt I is a minor toxin receptor inrodent motor neurons. FIG. 2B) Human Syt II differs from mouse/rat SytII by a single residue within the toxin binding site (residue 54 inmouse Syt II, 51 in human Syt II). FIG. 2C) Glutathione S-transferase(GST) tagged recombinant mouse Syt II 1-87 (m-Syt II) and a mouse Syt II1-87 mutant mimicking human Syt II (F54L, herein referred to as h-SytII) were immobilized on glutathione-Sepharose beads, and were used topull down BoNT/B, BoNT/D-C, or BoNT/G, with or without the presence ofganglioside (Gangl). All three toxins bind to m-Syt II 1-87, but noth-Syt II in the pull-down assays. FIG. 2D) Cultured rat hippocampalneurons only express Syt I but not Syt II ⁸. Therefore, knocking down(KD) Syt I generates neurons with no endogenous toxin receptors.Full-length m-Syt II and h-Syt II were then expressed in Syt I KDhippocampal neurons, and these neurons were exposed to BoNT/B (20 nM, 5min exposure, 24 hrs incubation). It has been found that h-Syt II wassignificantly less efficient than m-Syt II in mediating the entry ofBoNT/B, BoNT/D-C, and BoNT/G into Syt I KD neurons, as evidenced by thedegrees of cleavage of toxin substrate synaptobrevin (Syb). FIG. 2E) RatSyt I 1-83 and human Syt I 1-80 were used to pull down BoNT/B, BoNT/D-C,and BoNT/G, as described in panel C. Human Syt I mediated similar levelsof toxin binding as rat Syt I did for all three toxins. FIG. 2A to E areadapted from the recent publication: Peng et al, J. Cell Science, 2012,125:3233 ¹³. FIG. 2F) The binding affinity of BoNT/B (also defined asBoNT/B1) and one of its subtypes known as BoNT/B2 to rat Syt II aredetermined in an competition assay, by using the receptor binding domainof BoNT/B1 and B2 (right panel) to compete the binding of ¹²⁵I labeledBoNT/B1 on recombinant Syt II (left panel). The IC50 (which reflect thebinding affinity) is 0.48 nM for BoNT/B1, and 2 nM for BoNT/B2, 4-folddifference. This affinity difference is due to the C-terminal of thereceptor binding domain (residue 1028-1291), because exchanging thisregion between BoNT/B1 and BoNT/B2 (right panel) virtually switchestheir binding affinity (right panel). FIG. 2G) List of residues that aredifferent between BoNT/B1 and BoNT/B2. These residues are thought to bethe reason for the binding affinity difference between these two toxinsto rodent Syt II. Therefore, these may be key residues that caninfluence the binding affinity between BoNT/B and human Syt II. Panels Fto G are adapted from Ihara et al, 2003, BBA, 1625:19 ²⁹. (H) Singleresidue mutations within the receptor binding domain of BoNT/A andBoNT/B, as indicated in the table, can significantly change the potencyand toxicity of these toxins. This panel is adapted from Rummel et al,2004, Mol. Microbiology, 51:631 ³⁰. (I) The co-crystal structure ofBoNT/B (grey) binding to Syt II (red) reveals the key residues (listedin the right table) that form the binding pocket in BoNT/B. This panelis adapted from Jin et al, 2006, Nature, 444:1092 ³²and Chai et al,2006, Nature, 444:1096 ³¹.

FIG. 3A-FIG. 3B shows targeted mutagenesis of BoNT/B-H_(C) and theireffects on binding to m-Syt II and h-Syt II. FIG. 3A) WT BoNT/B-H_(C)and indicated BoNT/B-H_(C) mutants were expressed as recombinantproteins in E. Coli. Bacterial lysates were harvested and incubated withimmobilized m-Syt II (1-87) or h-Syt II (1-87). Bound pellets wereanalyzed by immunoblot assays, detecting BoNT/B-H_(C) using the HAantibody. “Input” represents bacterial lysates. Mutants that show strongbinding to h-Syt II are indicated by arrows. FIG. 3B) A table thatcategorizes BoNT/B-H_(C) mutations tested in FIG. 3A.

FIG. 4A-FIG. 4B shows further characterization of selected BoNT/B-H_(C)mutants for their binding to Syt I and Syt II. FIG. 4A) BoNT/B-H_(C) WTand indicated mutants were expressed in E. Coli. Harvested bacteriallysates were incubated with immobilized GST-tagged human Syt I (1-80),with or without the presence of gangliosides. Bound materials wereanalyzed by immunoblot assays detecting BoNT/B-H_(C). E1191Msignificantly enhanced binding of BoNT/B-H_(C) to human Syt I, whereasV1118M has reduced binding to human Syt I than WT BoNT/B-H_(C). FIG. 4B)WT BoNT/B-H_(C) and E1191M mutant were purified as His6-taggedrecombinant proteins and were incubated with immobilized GST-taggedm-Syt II (1-87) or h-Syt II (1-87), with or without the presence oflipid co-receptor gangliosides (Gangl). BoNT/B-H_(C) cannot bind toh-Syt II without gangliosides and only displays a weak binding in thepresence of gangliosides. Purified E1191M mutant binds h-Syt II withoutgangliosides, and the binding is further enhanced in the presence ofgangliosides.

FIG. 5A-FIG. 5B show that binding to human Syt I/II can be furtherenhanced by combining selected single residue substitutions. FIG. 5A)Selected double mutants that combine two mutation sites as indicatedwere tested for their ability to bind m-Syt II and h-Syt II in pull-downassays as described in FIG. 3A. Combinations of two sites, E1191M orE1191Q with S1199L or S1199Y or S1199F (marked by arrows) displayedrobust binding to h-Syt II. FIG. 5B) Binding of selected double mutantsto human Syt I was analyzed in pull-down assays. All double mutantsdisplayed significantly enhanced binding to human Syt I as compared toWT BoNT/B-H_(C).

FIG. 6A-FIG. 6D show further characterization of a representative doublemutant, E1191M/S1199Y. FIG. 6A) BoNT/B-H_(C) WT, E1191M, andE1191M/S1199Y mutants were expressed in E. Coli and purified asHis6-tagged recombinant proteins. Equal amounts of these proteins (100nM) were incubated with immobilized GST-tagged m-Syt II (1-87) or h-SytII (1-87) as indicated, with or without the presence of gangliosides(Gangl). Bound materials were subjected to immunoblot analysis. “Input”represents the purified recombinant proteins in following orders: WT,E1191M, E1191M/S1199Y. WT BoNT/B-H_(C) cannot bind to h-Syt II withoutgangliosides and only displays a weak binding in the presence ofgangliosides (lane 4, 5). E1191M mutant binds h-Syt II withoutgangliosides, and binding is further enhanced in the presence ofgangliosides (lane 6,7). E1191M/S1199Y significantly enhanced binding toh-Syt II as compared to E1191M (lane 8, 9). Binding of E1191M/S1199Y toboth h-syt II (lane 8,9) and m-Syt II (lane 10,11) are at similar levelsas WT BoNT/B-HC binding to m-Syt II (lane 13,14). FIG. 6B) Equal amountsof BoNT/B-H_(C) WT, E1191M, and E1191M/S1199Y mutants were incubatedwith GST tagged h-Syt I. Bound materials were subjected to immunoblotanalysis. E1191M and E1191M/S1199Y both significantly enhanced bindingto h-Syt I as compared to WT BoNT/B-H_(C). FIG. 6C) Titrations (nM) ofpurified WT BoNT/B-HC were incubated with m-Syt II, while titrations ofpurified E1191M/S1199Y were incubated with h-Syt II, as indicated. Boundmaterials were subjected to immunoblot analysis. Binding ofE1191M/S1199Y to h-Syt II is at similar levels as binding of WTBoNT/B-H_(C) to m-Syt II. FIG. 6D) Binding affinity betweenE1191M/S1199Y and h-Syt II was estimated based on quantifying theimmunoblot results obtained in panel C. The Kd is estimated to be 19+/−3nM for E1191M/51199Y binding to h-Syt II, whereas the Kd for WT BoNT/Bbinding to m-Syt II is 68+/−12 nM. Therefore, binding of E1191M/S1199Yto h-Syt II is ˜3.5 fold higher than WT BoNT/B binding to m-Syt II.

FIG. 7 shows that BoNT/B-H_(C) E1191M/S1199Y mutant can bind to h-Syt IIexpressed on surface of neurons. Cultured rat hippocampal neuronsexpress only Syt I, but not Syt II. Therefore, knocking-down (KD) Syt Iexpression via lentiviral infection created neurons without anyendogenous Syt and that abolished the binding of WT and E1191M/S1199YBoNT/B-H_(C) (the second frame from the left). M-Syt II, m-Syt II(F54L), and h-Syt II were then expressed in these neurons via lentiviralinfection. WT BoNT/B-H_(C) can bind to m-Syt II, but not m-Syt II (F54L)or h-Syt II. E1191M/S1199Y mutant can bind to both m-Syt II and h-Syt IIon neuron surface. Synapsin was also labeled as a marker for synapses.

FIG. 8 is the amino acid sequence of the BoNT/B-Hc (strain 1; BoNT/B1Okra strain). Residues 857-1291 of BoNT/B, strain 1, GenBank:AB232927.1, (SEQ ID NO: 1).

FIG. 9 is the nucleic acid sequence encoding BoNT/B-Hc (strain B1, Okrastrain) residues 857-1291 of BoNT/B, strain 1, based on GenBank:AB232927.1), which has been optimized for expression in E. coli. Thenucleic acid sequence is shown in SEQ ID NO: 2.

FIG. 10 shows the amino acid sequence of C. botulinum serotype A (1296a.a.) (SEQ ID NO: 3).

FIG. 11 shows the amio acid sequence of C. botulinum serotype B (1291a.a.) (SEQ ID NO: 4).

FIG. 12 shows the amino acid sequence of C. botulinum serotype C1 (1291a.a.) (SEQ ID NO: 5).

FIG. 13 shows the amino acid sequence of C. botulinum serotype D (1276a.a.) (SEQ ID NO: 6).

FIG. 14 shows the amino acid sequence of C. botulinum serotype E (1252a.a.)(SEQ ID NO: 7).

FIG. 15 shows the amino acid sequence of C. botulinum serotype F (1274a.a.) (SEQ ID NO: 8).

FIG. 16 shows the amino acid sequence of C. botulinum serotype G (1297a.a.) (SEQ ID NO: 9).

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to the generation of C. botulinumneurotoxin (BoNT) polypeptide which has improved binding to its humanreceptors through the incorporation of a modified receptor bindingdomain. From these findings, a new generation of therapeutic BoNTs canbe created by utilizing the modified receptor binding domain identifiedherein, with improved efficacy and specificity to target human neuronsthan the currently utilized WT BoNTs.

Definitions

As used herein, the term “binding affinity” means how strong amolecule's binding activity is for a particular receptor system. Ingeneral, high binding affinity results from greater intermolecular forcebetween a binding domain and its receptor system while low bindingaffinity involves less intermolecular force between the ligand and itsreceptor. High binding affinity involves a longer residence time for thebinding domain at its receptor binding site than is the case for lowbinding affinity. As such, a molecule with a high binding affinity meansa lower concentration of that molecule is required to maximally occupythe binding sites of a receptor system and trigger a physiologicalresponse. Conversely, low binding affinity means a relatively highconcentration of a molecule is required before the receptor bindingsites of a receptor system is maximally occupied and the maximumphysiological response is achieved. Thus, a botulinum neurotoxin of thepresent invention with increased binding activity due to high bindingaffinity will allow administration of reduced doses of the toxin,thereby reducing or preventing unwanted side-effects associated withtoxin dispersal into non-targeted areas.

As the term is used herein, “significantly enhanced binding” when usedto describe the binding affinity of a C. botulinum neurotoxin moleculeof the present invention to a specific receptor, refers to an increasein binding affinity for a specific receptor that is substantiallyincreased (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe binding affinity of the wild type molecule) as compared to thenon-substituted version of the molecule. In one embodiment, the enhancedbinding is an order of magnitude or more higher than the Kd of thenon-substituted neurotoxin (e.g., the neurotoxin with a naturallyoccurring BoNT H_(C) molecule). The term “significantly enhancedbinding” when used to describe the binding affinity of a BoNT/B-H_(C)binding fragment produced by the point mutations described herein refersto an increase in binding affinity of the modified binding domain(expressed as an isolated fragment of the entire BoNT protein) to aspecific receptor that is substantially increased (e.g., by 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% of the binding affinity) ascompared to the binding of the non-substituted version of the molecule.In one embodiment, the enhanced binding is significantly higher (e.g.,1.5×, 2.0×, 2.5×, 3.0×, etc.) than the Kd of the non-substitutedfragment.

As used herein, the term “botulinum neurotoxin” means any polypeptidethat can execute the overall cellular mechanism whereby a C. botulinumtoxin enters a neuron and inhibits neurotransmitter release andencompasses the binding of a C. botulinum toxin to a low or highaffinity receptor complex, the internalization of the toxin, thetranslocation of the toxin light chain into the cytoplasm and theenzymatic modification of a C. botulinum toxin substrate.

A “modified receptor binding domain” or “modified H_(e)”, as the term isused herein, facilitates the binding of the C. botulinum neurotoxinmolecule in which it is comprised, to a receptor for C. botulinumneurotoxin located on the surface of a target cell. Such a molecule istypically generated through genetic recombination technology. Themodified H_(C) has a binding activity for the receptor for C. botulinumneurotoxin located on the surface of a target cell. As used herein, theterm “binding activity” means that one molecule is directly orindirectly contacting another molecule via at least one intermolecularor intramolecular force, including, without limitation, a covalent bond,an ionic bond, a metallic bond, a hydrogen bond, a hydrophobicinteraction, a van der Waals interaction, and the like, or anycombination thereof. “Bound” and “bind” are considered terms forbinding.

As used herein, the term “C. botulinum toxin protease domain” means a C.botulinum toxin domain that can execute the enzymatic targetmodification step of the intoxication process. Thus, a C. botulinumtoxin protease domain specifically targets a C. botulinum toxinsubstrate and encompasses the proteolytic cleavage of a C. botulinumtoxin substrate, such as, e.g., SNARE proteins like a SNAP-25 substrate,a VAMP substrate and a Syntaxin substrate.

Non-limiting examples of C. botulinum toxin protease domains areprovided in Table 1 and 2.

As used herein, the term “C. botulinum toxin translocation domain” or“H_(N)” means a C. botulinum toxin domain that can execute thetranslocation step of the intoxication process that mediates C.botulinum toxin light chain translocation. Thus, a H_(N) facilitates themovement of a C. botulinum toxin light chain across a membrane andencompasses the movement of a C. botulinum toxin light chain through themembrane an intracellular vesicle into the cytoplasm of a cell.Non-limiting examples of a H_(N) include a BoNT/A H_(N), a BoNT/B H_(N),a BoNT/C1 H_(N), a BoNT/D H_(N), a BoNT/E H_(N), a BoNT/F H_(N), and aBoNT/G H_(N), the amino acid sequences of which are provided in Table 1and FIGS. 10-16.

As used herein, the term “C. botulinum receptor-binding domain” issynonomous with “He domain” and means any naturally occurring C.botulinum receptor binding domain that can execute the cell binding stepof the intoxication process, including, e.g., the binding of the C.botulinum toxin to a C. botulinum toxin-specific receptor system locatedon the plasma membrane surface of a target cell. It is envisioned thatreplacement of the binding activity can be achieved by, e.g., replacingthe entire C. botulinum H_(C) domain with a modified (e.g., enhanced)H_(C) domain.

As used herein, the term “C. botulinum toxin target cell” means a cellthat is a naturally occurring cell that a naturally occurring C.botulinum toxin is capable of intoxicating, including, withoutlimitation, motor neurons; sensory neurons; autonomic neurons; such as,e.g., sympathetic neurons and parasympathetic neurons; non-petidergicneurons, such as, e.g., cholinergic neurons, adrenergic neurons,noradrenergic neurons, serotonergic neurons, GABAergic neurons; andpeptidergic neurons, such as, e.g., Substance P neurons, Calcitonin GeneRelated Peptide neurons, vasoactive intestinal peptide neurons,Neuropeptide Y neurons, cholecystokinin neurons.

By “isolated” is meant a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings, e.g. from flanking DNA or from the natural source of theDNA.

The term “purified” is used to refer to a substance such as apolypeptide that is “substantially pure”, with respect to othercomponents of a preparation (e.g., other polyepeptides). It can refer toa polypeptide that is at least about 50%, 60%, 70%, or 75%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% pure, with respect to other components.Recast, the terms “substantially pure” or “essentially purified”, withregard to a polypeptide, refers to a preparation that contains fewerthan about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, mostpreferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of oneor more other components (e.g., other polypeptides or cellularcomponents).

The term “conservative” or “conservative substitution mutation” as usedherein refers to a mutation where an amino acid is substituted foranother amino acid that has similar properties, such that one skilled inthe art of peptide chemistry would expect the secondary structure,chemical properties, and/or hydropathic nature of the polypeptide to besubstantially unchanged. The following groups of amino acids have beenhistorically substituted for one another as conservative changes: (1)ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, try, thr; (3)val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp,his. Other commonly accepted conservative substitutions are listedbelow:

Residue Conservative Substitutions Residue Conservative SubstitutionsAla Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile AspGlu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu AspTrp Tyr Gly Pro Tyr Trp; Phe His Asn; Gin Val Ile; Leu Ile Leu, Val

The term “substitution mutation” without the reference to a specificamino acid, may include any amino acid other than the wild type residuenormally found at that position. Such substitutions may be replacementwith non-polar (hydrophobic)) amino acids, such as glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, andproline. Substitutions may be replacement with polar (hydrophyllic)amino acids such as serine, threonine, cysteine, tyrosine, asparagine,and glutamine. Substitutions may be replacement with electricallycharged amino acids e.g., negatively electrically charged amino acidssuch as aspartic acid and glutamic acid and positively electricallycharged amino acids such as lysine, arginine, and histidine.

The substitution mutations described herein will typically bereplacement with a different naturally occurring amino acid residue, butin some cases non-naturally occurring amino acid residues may also besubstituted. Non-natural amino acids, as the term is used herein, arenon-proteinogenic (i.e., non-protein coding) amino acids that eitheroccur naturally or are chemically synthesized. Examples include but arenot limited to β-amino acids (β3 and β2), homo-amino acids, proline andpyruvic acid derivatives, 3-substituted alanine derivatives, glycinederivatives, ring-substituted phenylalanine and tyrosine derivatives,linear core amino acids, diamino acids, D-amino acids, and N-methylamino acids. In some embodiments, the amino acid can be substituted orunsubstituted. The substituted amino acid or substituent can be ahalogenated aromatic or aliphatic amino acid, a halogenated aliphatic oraromatic modification on the hydrophobic side chain, or an aliphatic oraromatic modification.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically significant reduction in one ormore symptoms of the condition when administered to a typical subjectwho has the condition. A therapeutically significant reduction in asymptom is, e.g. about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 100%, or more ascompared to a control or non-treated subject.

The term “treat” or “treatment” refers to therapeutic treatment whereinthe object is to eliminate or lessen symptoms. Beneficial or desiredclinical results include, but are not limited to, elimination ofsymptoms, alleviation of symptoms, diminishment of extent of condition,stabilized (i.e., not worsening) state of condition, delay or slowing ofprogression of the condition.

As used herein, a “subject” refers to a human or animal. Usually theanimal is a vertebrate such as a primate, rodent, domestic animal orgame animal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female. A subject can be a fully developedsubject (e.g., an adult) or a subject undergoing the developmentalprocess (e.g., a child, infant or fetus).

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with unwanted neuronal activity. In addition, themethods and compositions described herein can be used to treatdomesticated animals and/or pets.

Embodiments

The observation that BoNT/B is less specific and potent in humans due toits inability to bind human Syt II, may explain why comparatively higherdoses are required than BoNT/A. Higher BoNT/B doses correspond toincreased chances for triggering antibody responses and for seriousside-effects to occur. Therefore, improved binding of BoNT/B to thehuman receptor Syt II, to increase its efficacy and specificity totarget human neurons should allow a reduced amount of the toxin dosesused in therapeutic applications.

Aspects of the invention arise from the finding that modifying theprotein sequence of BoNT/B-H_(C) modifies binding of the fragmentcontaining the receptor binding domain, to the human Syt II receptor.Specific modifications have been identified that enhance binding,thereby generating a domain that binds human Syt II with high-affinity.The modified BoNT/B-H_(C), when in the context of a full length BoNTprotein, retains these binding properties. Incorporation of a modifiedreceptor binding domain with enhanced binding, into a moleculecomprising the other BoNT domains, thereby generates a full length BoNTmolecule with similarly enhanced receptor bindings. As such, newversions of BoNT with high-affinity binding to human Syt II aregenerated. BoNT with significantly enhanced binding can be used insimilar therapies, albeit at lower doses than presently available BoNTmolecules, thus providing safer methods of treatment.

The BoNT polypeptides, including full-length BoNT polypeptides and BoNTpolypeptide fragments or domains described herein, and nucleic acidmolecules which encode them, are explicitly encompassed in theinvention. These polypeptides and nucleic acid molecules can begenerated by recombinant DNA procedures known in the art. Suchpolypeptides are typically referred to as “recombinant polypeptides” or“recombinant nucleic acids”.

BoNT has the overall structure shown in FIG. 1B. BoNT is comprised ofthree domains, each domain having a specific and independent function: aprotease domain (also referred to as the light chain), a translocationdomain (H_(N)), and a receptor-binding domain (H_(C)). Domains of thevarious strains of C. botulinum neurotoxin have been shown to be largelyinterchangeable (as demonstrated by naturally occurred chimeric toxinssuch as BoNT/CD, which is composed of the light chain and H_(N) ofBoNT/C, with the H_(C) of BoNT/D ³⁴, in U.S. Pat. No. 8,052,979). Theprotein can be in single chain form or di-chain form. The di-chain formresults from the naturally occurring protease processing of a proteasecleavage site located between the protease domain and the translocationdomain. The protein is maintained in the Di-chain form followingprotease processing by the presence of a di-sulfide bond.

One aspect of the invention relates to a botulinum neurotoxin (BoNT)comprising a protease domain, a translocation domain, and a modifiedreceptor binding domain of Clostridial botulinum serotype B, asdescribed herein, and a protease cleavage site. Typically these arearranged in a linear amino-to-carboxyl single polypeptide order of theprotease domain, the protease cleavage site, the translocation domainand the modified receptor binding domain. However, differentarrangements of the various domains are expected to function adequately.In one embodiment, the modified receptor binding domain comprises one ormore substitution mutations which lead to significantly enhanced bindingto the human Syt I receptor and/or the human Syt II receptor.

Strains of Clostridia botulinum produce seven antigenically-distincttypes of Botulinum toxins (BoNTs), which have been identified byinvestigating botulism outbreaks in man (BoNT/A, /B, /E and /F), animals(BoNT/C1 and /D), or isolated from soil (BoNT/G). While all seven BoNTserotypes have similar structure and pharmacological properties, eachalso displays heterogeneous bacteriological characteristics. The geneticdiversity of the C. botulinum strains is described in detail in Hill etal. (Journal of Bacteriology, Vol. 189, No. 3, p. 818-832 (2007)) ³⁵,the contents of which are incorporated herein by reference.

Toxins from the various C. botulinum strains share the same functionaldomain organization and overall structural architecture. C. botulinumtoxins are each translated as a single chain polypeptide ofapproximately 150 kDa that is subsequently cleaved by proteolyticscission within a disulfide loop by a naturally-occurring protease, suchas, e.g., an endogenous C. botulinum toxin protease or anaturally-occurring proteases produced in the environment. Thisposttranslational processing yields a di-chain molecule comprising anapproximately 50 kDa light chain (LC) and an approximately 100 kDa heavychain (HC) held together by a single disulfide bond and noncovalentinteractions. Each mature di-chain molecule comprises three functionallydistinct domains: 1) a proteolytic domain located in the LC thatincludes a metalloprotease region containing a zinc-dependentendopeptidase activity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the LC from intracellular vesicles into the cytoplasm of thetarget cell; and 3) a binding domain found within the carboxyl-terminalhalf of the HC that determines the binding activity and bindingspecificity of the toxin to the receptor complex located at the surfaceof the target cell. The locations of the specific domains within thetoxin are provided in Table 1:

TABLE 1 C. botulinum toxin domains from various strains Toxin LC H_(N)H_(C) BoNT/A M1-K448 A449-K871 N872-L1296 BoNT/B M1-K441 A442-S858E859-E1291 BoNT/C1 M1-K449 T450-N866 N867-E1291 BoNT/D M1-R445 D446-N862S863-E1276 BoNT/E M1-R422 K423-K845 R846-K1252 BoNT/F M1-K439 A440-K864K865-E1274 BoNT/G M1-K446 S447-S863 N864-E1297

Complete amino acid sequences of the toxins are provided in FIGS. 10-16.

The binding, translocation and protease activity of these threefunctional domains are all necessary for toxicity. The overall cellularintoxication mechanism whereby C. botulinum toxins enter a neuron andinhibit neurotransmitter release is similar, regardless of serotype orsubtype. Without wishing to be bound by theory, the intoxicationmechanism involves at least four steps: 1) receptor binding, 2) complexinternalization, 3) light chain translocation, and 4) protease targetmodification. The process is initiated when the H_(c) domain of a C.botulinum toxin binds to a toxin-specific receptor located on the plasmamembrane surface of a target cell. The binding specificity of a receptorcomplex is thought to be achieved, in part, by specific combinations ofgangliosides and protein receptors. Once bound, the toxin/receptorcomplexes are internalized by endocytosis and the internalized vesiclesare sorted to specific intracellular routes. The translocation step istriggered by the acidification of the vesicle compartment. Oncetranslocated, light chain endopeptidase of the toxin is released fromthe intracellular vesicle into the cytosol where it specifically targetsone of three proteins known as the core components of theneurotransmitter release apparatus (vesicle-associated membrane protein(VAMP)/synaptobrevin, synaptosomal-associated protein of 25 kDa(SNAP-25) and Syntaxin). These core components are necessary forsynaptic vesicle docking and fusion at the nerve terminal and constitutemembers of the soluble N-ethylmaleimide-sensitive factor-attachmentprotein-receptor (SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in thecarboxyl-terminal region, releasing a nine or twenty-six amino acidsegment, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic plasma membranesurface. The selective proteolysis of synaptic SNAREs accounts for theblock of neurotransmitter release caused by C. botulinum toxins in vivo.The SNARE protein targets of C. botulinum toxins are common toexocytosis in a variety of non-neuronal types; in these cells, as inneurons, light chain peptidase activity inhibits exocytosis, see, e.g.,Yann Humeau et al., How Botulinum and Tetanus Neurotoxins BlockNeurotransmitter Release, 82(5) Biochimie. 427-446 (2000); KathrynTurton et al., Botulinum and Tetanus Neurotoxins: Structure, Functionand Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxinsin Neurons, 11(9) Trends Microbiol. 431-437, (2003).

The botulinum neurotoxin of the present invention comprises a modifiedreceptor binding domain. The modified receptor binding domain exhibitssignificantly enhanced binding to one or more human receptors typicallybound and utilized by one or more C. botulinum toxin strains. Examplesof specific modified receptor binding domains are provided herein. Theisolated modified receptor binding domain polypeptide described hereinis also encompassed by the present invention, as is the isolated nucleicacid molecule by which it is encoded.

The botulinum neurotoxin of the present invention also comprises aprotease domain, also referred to in the art as a light chain variant.The light chain variant may be a naturally occurring light chainvariant, such as, e.g., C. botulinum toxin light chain isoforms and C.botulinum toxin light chain subtypes; or a non-naturally occurring C.botulinum toxin light chain variant, such as, e.g., conservativesubstitution C. botulinum toxin light chain variants.

The botulinum neurotoxin of the present invention also comprises a toxintranslocation domain (H_(N)).

The various domains described herein (e.g., H_(N), H_(c), or proteasedomain) include, without limitation, naturally occurring variants, suchas, e.g., isoforms and subtypes; non-naturally occurring variants, suchas, e.g., conservative substitution mutations. Non-naturally-occurringvariants, refers to a domain that has at least one amino acid changefrom the corresponding region of the reference sequences (e.g., fromTable 1 or FIGS. 10-16) and can be described in percent identity to thecorresponding region of that reference sequence.

It is recognized by those of skill in the art that within each serotypeof C. botulinum toxin there can be naturally occurring C. botulinumdomain variants that differ somewhat in their amino acid sequence, andalso in the nucleic acids encoding these proteins. A naturally occurringC. botulinum toxin domain (e.g., light chain, H_(N) or H_(c)) variantenvisioned for use in the generation of the BoNT of the presentinvention can function in substantially the same manner as the referenceC. botulinum toxin domain on which the naturally occurring C. botulinumdomain variant is based, and can be substituted for the reference C.botulinum toxin domain in any aspect of the present invention.

A non-limiting example of a naturally occurring C. botulinum toxindomain variant is a C. botulinum toxin domain isoform such as, e.g., aBoNT/A domain isoform, a BoNT/B domain isoform, a BoNT/C 1 domainisoform, a BoNT/D domain isoform, a BoNT/E domain isoform, a BoNT/Fdomain isoform, and a BoNT/G domain isoform. A C. botulinum toxin domainisoform can function in substantially the same manner as the referenceC. botulinum toxin domain on which the C. botulinum toxin domain isoformis based, and can be substituted for the reference C. botulinum toxindomain in any aspect of the present invention.

Another non-limiting example of a naturally occurring C. botulinum toxindomain variant is a C. botulinum toxin domain subtype such as, e.g., adomain from subtype BoNT/A1, BoNT/A2,BoNT/A3, BoNT/A4, BoNT/A5; a domainfrom subtype BoNT/B1, BoNT/B2, BoNT/B3, BoNT/B4, BoNT/B5, BoNT/B6,BoNT/B7; a domain from subtype BoNT/C1-1, BoNT/C1-2, BoNT/D-C; a domainfrom subtype BoNT/E1, BoNT/E2, BoNT/E3, BoNT/E4, BoNT/E5, BoNT/E6,BoNT/E7, BoNT/E8; and a domain from subtype BoNT/F1, BoNT/F2, BoNT/F3,BoNT/F4, BoNT/F5, BoNT/F6, BoNT/F7. A C. botulinum toxin domain subtypecan function in substantially the same manner as the reference C.botulinum toxin domain on which the C. botulinum toxin domain subtype isbased, and can be substituted for the reference C. botulinum toxindomain in any aspect of the present invention.

As used herein, the term “non-naturally occurring variant” (e.g., C.botulinum toxin light chain variant, H_(C) and H_(N)) means a C.botulinum domain produced with the aid of human manipulation, including,without limitation, domains produced by genetic engineering using randommutagenesis or rational design and C. botulinum domains produced bychemical synthesis. Non-limiting examples of non-naturally occurring C.botulinum domain variants include, e.g., conservative C. botulinumdomain variants. As used herein, the term “conservative C. botulinumdomain variant” means a C. botulinum domain that has at least one aminoacid substituted by another amino acid or an amino acid analog that hasat least one property similar to that of the original amino acid fromthe reference C. botulinum domain sequence (e.g., Table 1 and FIGS.10-16). The variant may have one, two, three, four, five or moreconservative amino acid substitutions compared to the reference domainsequence. Examples of properties include, without limitation, similarsize, topography, charge, hydrophobicity, hydrophilicity, lipophilicity,covalent-bonding capacity, hydrogen-bonding capacity, a physicochemicalproperty, of the like, or any combination thereof. A conservative C.botulinum domain variant can function in substantially the same manneras the reference C. botulinum toxin domain on which the conservative C.botulinum toxin domain variant is based, and can be substituted for thereference C. botulinum domain in any aspect of the present invention.

A non-naturally occurring C. botulinum toxin domain variant maysubstitute one or more amino acids (e.g., one, two, three, four, five ormore) from the reference C. botulinum toxin domain on which thenaturally occurring C. botulinum toxin domain is based. A non-naturallyoccurring C. botulinum toxin domain variant can also possess 95% or more(e.g., 96%, 97%, 98% or 99%) amino acid identity to the reference C.botulinum toxin domain on which the naturally occurring C. botulinumdomain variant is based.

Various non-naturally occurring C. botulinum neurotoxins or specificdomains thereof, are described in International Patent PublicationsWO95/32738, WO96/33273, WO98/07864 and WO99/17806, each of which isincorporated herein by reference.

The C. botulinum neurotoxin or specific domain thereof described hereinwill typically contain naturally occurring amino acid residues, but insome cases non-naturally occurring amino acid residues may also bepresent. Therefore, so-called “peptide mimetics” and “peptideanalogues”, which may include non-amino acid chemical structures thatmimic the structure of a particular amino acid or peptide, may also beused within the context of the invention. Such mimetics or analogues arecharacterised generally as exhibiting similar physical characteristicssuch as size, charge or hydrophobicity, and the appropriate spatialorientation that is found in their natural peptide counterparts. Aspecific example of a peptide mimetic compound is a compound in whichthe amide bond between one or more of the amino acids is replaced by,for example, a carbon-carbon bond or other non-amide bond, as is wellknown in the art (see, for example Sawyer, in Peptide Based Drug Design,pp. 378-422, ACS, Washington D.C. 1995).

In one aspect of the invention, the botulinum neurotoxin (BoNT) of thepresent invention comprises a modified receptor binding domain of C.botulinum serotype B (BoNT/B-H_(C)). The modified BoNT/B-H_(C) comprisesone or more substitution mutations which lead to significantly enhancedbinding to the human Syt I receptor and/or the human Syt II receptor. Inone embodiment, the BoNT/B-H_(c) is from BoNT/B1 (GenBank access No.:AB232927.1). The amino acid sequence of BoNT/B1-H_(C) Okra strain, usedas the reference template in the present invention is shown in FIG. 8.The generation of B-H_(c) from other strains by substitution of theamino acids that correspond to the specified position(s) in B1 describedherein is also envisioned. Also encompassed in the invention is anisolated, purified modified receptor binding domain polypeptidedescribed herein. The present invention also encompasses a polypeptidecomprising a modified receptor binding domain described herein. Theinvention also encompasses a nucleic acid molecule which encodes such apolypeptide. In one embodiment, the modified receptor binding domain isBoNT/B-H_(C) (e.g., from BoNT/B1).

Modification of the BoNT/B-H_(C) protein sequence can be performed byeither targeted mutagenesis (site-directed mutagenesis) or randommutagenesis of each amino acid residue within the region known forbinding Syt I/II. These Syt binding regions are well defined by previousstudies relating to mouse or rate Syt receptors ^(1,29,3631,32) but havenot been clearly determined for interactions between BoNT/B-H_(c) andhuman Syt receptors. Different subtypes of BoNT/B can be used as thetemplate to create the same or similar mutations by generatingcorresponding mutations described herein for B1-H_(C). The correspondingposition for selected residues to be mutated can be readily identifiedby sequence alignment with the B1 subtype. The resulting polypeptideproducts are encompasses by the instant invention, as are polypeptidescomprising said products and nucleic acid molecules encoding saidpolypeptides and products.

Amino acid sequence modifications to produce the modified receptorbinding domain can be mutation of a single residue to a different aminoacid (single site substitution), mutation of multiple residues at thesame time (multiple sites substitution), deletion of one or moreresidues (deletion), and insertion of one or more residues (insertion),as well as combinations thereof. Methods for mutating proteins arewell-known in the art (e.g., targeted single site and multiple sitessubstitutions on the DNA encoding the BoNT/B-H_(C) sequence).

In one embodiment, one or more residues in BoNT/B-H_(C) that eithercontact rodent Syt II or the surrounding regions, based on previousliteratures on BoNT/B receptor binding domain ²⁹ and reported BoNT/B-SytII structure (PDB ID: 2NM1) ^(31,32), are modified. These include,without limitation those positions that correspond to position Y1181,P1197, A1196, F1204, F1194, P1117, W1178, Y1183, V1118, 51116, K1113,K1192, S1199, S1201, E1191, E1245, Y1256 of BoNT/B-B1. In oneembodiment, one or more of these residues is modified to a hydrophobicamino acid (e.g., V, I, L, M, F, W, C). In one embodiment, one or moreof these residues is modified to a less hydrophobic amino acid (e.g., A,Y, H, T, S, P, Q, N and G). Combinations of various modifications arealso envisioned, including, without limitation, mutations of two or morerecited positions, to any variety of the herein recited various aminoacids.

In one embodiment, the BoNT/B-H_(C) has one or more substitutionmutation (e.g., at positions which correspond to positions E1191, S1199,S1201, V1118, P1117, Y1183, A1196, and Y1181 of B1) that enhancesbinding to human Syt II as compared to WT BoNT/B-H_(C). In oneembodiment, the mutation comprises one or more mutations that correspondto E1191M/I/T/L/Q (E1191M, E1191I, E1191T, E1191L, or E1191Q), V1118M,S1199Y/L/F (S1199Y, S1199L, or S1199F), S1201V, P1117S/M/Y (P1117S,P1117M, or P1117Y), Y1183M, Y1181M, A1196Y of B1, or combinationsthereof (FIG. 3A, B). Suitably the mutations are selected form the abovemutations at positions 1118, 1191 and 1199 or combinations thereof. Inparticular, mutations selected from one or more of V1118M, E1191M/Q/Iand S1199Y may be beneficial. More particularly, the mutation thatcorresponds to position E1191M or E1191Q of B1 is envisioned, since theydisplay the strongest enhancement for binding h-Syt II. The mutationscorresponding to E1191M or E1191Q of B1 also significantly enhancedbinding of BoNT/B-H_(C) to human Syt I as compared to WT BoNT/B-H_(C)(FIG. 4A). In one embodiment, the BoNT/B-H_(C) has two substitutionmutations.

Multiple site substitutions can also be generated by combining mutationsin these identified key residues. Such multiple site substitutionmutants have further enhanced binding to human Syt I and h-Syt II (FIG.5). As a non-limiting example, mutations that combine two single sitesubstitutions such as those corresponding to E1191M or E1191Q withS1199L, S1199Y or S1199F of B1 displayed significantly enhanced bindingto both human Syt I and h-Syt II (FIG. 5). The enhancement in bindingstrength was surprising given the relatively modest enhancement inbinding activity achieved by mutations at the 1199 position alone.

In one embodiment substitution of a residue corresponding to positionE1191, S1199, S1201, V1118, P1117, A1196, Y1181, and Y1183 of BoNT/B-B1is envisioned, since it will yield a BoNT/B-H_(C) mutant with enhancedbinding to human Syt II. Additional combination substitutions atpositions including, but not limited to those that correspond to E1191,S1199, S1201, V1118, P1117, Y1181, Y1183, and A1196 of B1 yieldBoNT/B-H_(C) mutants with enhanced binding to human Syt II.

Accordingly, the invention encompasses polypeptides comprisingBoNT/B-H_(C) with modified amino acid sequence relative to the sequenceof WT BoNT/B-H_(C), wherein the modified BoNT/B-H_(C) has significantlyenhanced binding to human Syt I and II as compared to WT BoNT/B-H_(C).The invention further encompasses nucleic acid molecules encoding suchpolypeptides. In a preferred embodiment, the modified BoNT/B-H_(C)mutants contain amino acids substitutions at one or combinations of theamino acid residues corresponding to V1118, E1191, S1199, S1201, P1117,Y1181, Y1183, and A1196 of B1. In one embodiment, these modificationsinclude mutations corresponding to E1191M or E1191Q in combination withS1199L, S1199Y or S1199F of B1.

The present invention also encompass mutant full-length BoNT/B thatcontain the same amino acid substitutions in B-H_(c) as described abovefor therapeutic applications in humans. In a preferred embodiment, thefull-length BoNT/B mutants contain amino acids substitutions at one orcombinations of the amino acid residues corresponding to position E1191,V1118, S1199, S1201, P1117, Y1181, Y1183, and A1196 of B1. In oneembodiment, the modifications include combinations of E1191M or E1191Qwith S1199L, S1199Y or S1199F. The mutations can be made in the samemanner as disclosed above for BoNT/B-H_(C), using any one of BoNT/Bsubtypes as templates. These mutant BoNT/B toxins have significantlyenhanced binding to both human Syt II and human Syt I, therefore willachieve higher efficacy and specificity to target human neurons than WTBoNT/B.

Toxin diffusion and generation of neutralization antibodies are notlimited to BoNT/B, but also observed for BoNT/A, indicating that thebinding affinity of BoNT/A to its receptor SV2 also needs to beimproved. Because BoNT/B binding to Syt I/II has much higher affinitythan BoNT/A binding to SV2 ^(14,20,26,27,) a modified BoNT/B receptorbinding domain (BoNT/B-H_(C)) with the ability to bind human Syt II canalso be used to replace BoNT/A-H_(C) to generate a modified chimericBoNT/A with greater efficacy and specificity for human neurons than WTBoNT/A. ²⁸ ²⁹ ³⁰

It is further envisioned that the modified BoNT/B-H_(C) described abovecan be utilized to replace the H_(C) of all other BoNTs. The H_(C)regions of each BoNTs are well defined and their replacement can beperformed via standard PCR fusion of DNA encoding BoNT/B-H_(C) with theH_(N)-LC of other BoNTs, which has been well-established in the art. Inaddition, these replacements may also be performed using the C-terminalpart of BoNT/B-H_(C) (designated as H_(CC)), which is the regioncontaining the binding site for protein receptors and gangliosides ineach BoNT. The resulting chimeric toxins will have the ability to targethuman neurons via binding to human Syt I/II. As a non-limiting example,modified BoNT/B-H_(C) can be used to replace the H_(C) of BoNT/A. Theresulting polypeptides are encompassed by the instant invention. Thesechimeric toxin will have a higher efficacy and specificity targetinghuman neurons than WT BoNT/A. Such a chimeric BoNT/A toxin can be usedfor therapeutic applications in humans and offers significantimprovements over WT BoNT/A.

Another aspect of the invention relates to an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes the polypeptidesdescribed herein (e.g., modified receptor binding domain or thebotulinum neurotoxin comprising the modified receptor binding domain,described herein). In one embodiment, the nucleic acid moleculecomprises the nucleic acid sequence shown in FIG. 9. Such nucleic acidmolecules can be produced by recombinant DNA techniques.

Another aspect of the invention relates to a nucleic acid vectorcomprising the nucleic acid molecule described herein. In one embodimentthe vector is an expression vector. Such an expression vector isreferred to herein as an expression construct, and comprises a nucleicacid molecule disclosed herein operably-linked to the expression vectoruseful for expressing the nucleic acid molecule in a cell or cell-freeextract. A wide variety of expression vectors can be employed forexpressing a nucleic acid molecule encoding a C. botulinum neurotoxin ofthe present invention including, without limitation, a viral expressionvector; a prokaryotic expression vector; eukaryotic expression vectors,such as, e.g., a yeast expression vector, an insect expression vectorand a mammalian expression vector; and a cell-free extract expressionvector. It is further understood that expression vectors useful topractice aspects of these methods may include those which express the C.botulinum neurotoxin under control of a constitutive, tissue-specific,cell-specific or inducible promoter element, enhancer element or both.Non-limiting examples of expression vectors, along with well-establishedreagents and conditions for making and using an expression constructfrom such expression vectors are readily available from commercialvendors that include, without limitation, BD Biosciences-Clontech, PaloAlto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN,Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection,making and use of an appropriate expression vector are routineprocedures well within the scope of one skilled in the art and from theteachings herein.

Another aspect of the invention relates to a cell comprising the nucleicacid molecule or expression construct described herein. The cell can befor propagation of the nucleic acid or for expression of the nucleicacid, or both. Such cells include, without limitation, prokaryotic cellsincluding, without limitation, strains of aerobic, microaerophilic,capnophilic, facultative, anaerobic, gram-negative and gram-positivebacterial cells such as those derived from, e.g., Escherichia coli,Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis,Clostridia perfringens, Clostridia difficile, Caulobacter crescentus,Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls,Neisseria meningitidis, Pseudomonas fluorescens and Salmonellatyphimurium; and eukaryotic cells including, without limitation, yeaststrains, such as, e.g., those derived from Pichia pastoris, Pichiamethanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomycescerevisiae and Yarrowia lipolytica; insect cells and cell lines derivedfrom insects, such as, e.g., those derived from Spodoptera frugiperda,Trichoplusia ni, Drosophila melanogaster and Manduca sexta; andmammalian cells and cell lines derived from mammalian cells, such as,e.g., those derived from mouse, rat, hamster, porcine, bovine, equine,primate and human. Cell lines may be obtained from the American TypeCulture Collection, European Collection of Cell Cultures and the GermanCollection of Microorganisms and Cell Cultures. Non-limiting examples ofspecific protocols for selecting, making and using an appropriate cellline are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F.A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES:FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer AcademicPublishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUESOF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUECULTURE: LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley andSons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUE (Wiley-Liss, 4.sup.th ed. 2000); ANIMAL CELL CULTURE: APRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press,3.sup.rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra,(2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, OxfordPress, 2.sup.nd ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,supra, (2004). These protocols are routine procedures within the scopeof one skilled in the art and from the teaching herein.

It is also envisioned that the modified BoNT/B-H_(C) described here canbe utilized as a delivery tool to target neurons in humans. For example,the modified BoNT/B-H_(C) can be linked to other therapeutic agents,covalently or non-covalently, and acts as the targeting vehicle todeliver the therapeutic agents to neurons in humans by binding to humanSyt I/II. As such, another aspect of the invention relates to a chimericpolypeptide molecule comprising a first portion that is a modifiedreceptor binding domain of C. botulinum serotype B, comprising one ormore substitution mutations which leads to significantly enhancedbinding to the human Syt I receptor and/or the human Syt II receptor,linked to a second portion. The second portion of the molecule can be abioactive molecule such as a therapeutic agent (e.g., a polypeptide ordrug). Linkage of the first and second portions of the molecule can becovalent (e.g., in the form of a fusion protein) or non-covalent.Methods of such linkage are known in the art and can readily be appliedby the skilled practitioner.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising the C. botulinum neurotoxin, or chimeric moleculedescribed herein. In one embodiment, the polypeptide described herein isan active ingredient in a composition comprising a pharmaceuticallyacceptable carrier (referred to herein as a pharmaceutical composition).A “pharmaceutically acceptable carrier” means any pharmaceuticallyacceptable means to mix and/or deliver the targeted delivery compositionto a subject. The term “pharmaceutically acceptable carrier” as usedherein means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject agents from one organ, or portion of the body, to another organ,or portion of the body. Each carrier must be “acceptable” in the senseof being compatible with the other ingredients of the composition and iscompatible with administration to a subject, for example a human. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, such as the routes of administrationdescribed herein. Supplementary active ingredients also can beincorporated into the compositions. When an agent, formulation orpharmaceutical composition described herein, is administered to asubject, preferably, a therapeutically effective amount is administered.As used herein, the term “therapeutically effective amount” refers to anamount that results in an improvement or remediation of the condition.In one embodiment, the pharmaceutical composition is formulated foradministration by injection. In one embodiment, the pharmaceuticalcomposition involves the botulinum neurotoxin encapsulated inmicrospheres. In one embodiment, the pharmaceutical composition involvesthe botulinum neurotoxin formulated for slow release.

In one embodiment, the botulinum neurotoxin, polypeptide, or chimericmolecule of the present invention is in the form of a controlled releaseformula. Such compositions and methods for adminstration are provides inU.S. Patent publication No. 2007/0020295, the contents of which areherein incorporated by reference.

Botulinum neurotoxin can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. The proteolytic strainsthat produce, for example, the botulinum toxin type B serotype may onlycleave a portion of the toxin produced. The exact proportion of nickedto unnicked molecules depends on the length of incubation and thetemperature of the culture. Therefore, a certain percentage of apreparation of, for example, the botulinum toxin type B toxin may beinactive. In one embodiment, the neurotoxin of the present invention isin an active state. In one embodiment, the neurotoxin is in an inactivestate. In one embodiment, a combination of active and inactiveneurotoxin is envisioned.

Also encompassed in the present invention is a kit comprising thepharmaceutical composition described herein. The kit may furthercomprise a delivery tool or device for the therapeutic administration ofthe composition, and/or instructions for therapeutic administration.

Another aspect of the invention relates to a delivery tool or device foradministration of the pharmaceutical compositions described herein,pre-loaded with the pharmaceutical composition (e.g., for single use).Such devices may be a syringe or a microneedle device for delivery ofthe compositions. The syringe may be a single use syringe pre-loadedwith an effective amount of the composition. The microneedle device maycomprise one or more microneedles coated with the composition describedherein, such as is described in U.S. Patent Publication 2010/0196445,the contents of which are incorporated herein in their entirety.

Methods of Treatment

The present invention also includes methods for treating a conditiontypically treated with a neurotoxin (e.g, skeletal muscle conditions,smooth muscle conditions, glandular conditions, a neuromusculardisorder, an autonomic disorder, pain, or an aesthetic/cosmeticcondition). Such conditions are associated with unwanted neuronalactivity, as determined by the skilled practitioner. The methodcomprises the step of administering a therapeutically effective amountof a pharmaceutical composition described herein (e.g., containing abotulinum neurotoxin (BoNT) or a chimeric molecule) to the appropriatelocation in the mammal to reduce the unwanted neuronal activity, tothereby treat the condition. Administration is by a route that contactsan effective amount of the composition to neurons exhibiting theunwanted activity.

Specific conditions envisioned for treatment by the methods discussedherein include, without limitation, spasmodic dysphonia, spasmodictorticollis, laryngeal dystonia, oromandibular dysphonia, lingualdystonia, cervical dystonia, focal hand dystonia, blepharospasm,strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focalspasticity and other voice disorders, spasmodic colitis, neurogenicbladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure,achalasia, dysphagia and other muscle tone disorders and other disorderscharacterized by involuntary movements of muscle groups, lacrimation,hyperhydrosis, excessive salivation, excessive gastrointestinalsecretions as well as other secretory disorders, pain from musclespasms, headache pain. In addition, the present invention can be used totreat dermatological or aesthetic/cosmetic conditions, for example,reduction of brow furrows, reduction of skin wrinkles The presentinvention can also be used in the treatment of sports injuries.

Borodic U.S. Pat. No. 5,053,005 discloses methods for treating juvenilespinal curvature, i.e. scoliosis, using botulinum type A. The disclosureof Borodic is incorporated in its entirety herein by reference. In oneembodiment, using substantially similar methods as disclosed by Borodic,a modified neurotoxin can be administered to a mammal, preferably ahuman, to treat spinal curvature. In a suitable embodiment, a modifiedneurotoxin comprising botulinum type E fused with a leucine-based motifis administered. Even more preferably, a modified neurotoxin comprisingbotulinum type A-E with a leucine-based motif fused to the carboxylterminal of its light chain is administered to the mammal, preferably ahuman, to treat spinal curvature.

In addition, the modified neurotoxin can be administered to treat otherneuromuscular disorders using well known techniques that are commonlyperformed with botulinum type A. For example, the present invention canbe used to treat pain, for example, headache pain, pain from musclespasms and various forms of inflammatory pain. For example, Aoki U.S.Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods ofusing botulinum toxin type A for treating pain. The disclosure of thesetwo patents is incorporated in its entirety herein by reference.

Autonomic nervous system disorders can also be treated with a modifiedneurotoxin. For example, glandular malfunctioning is an autonomicnervous system disorder. Glandular malfunctioning includes excessivesweating and excessive salivation. Respiratory malfunctioning is anotherexample of an autonomic nervous system disorder. Respiratorymalfunctioning includes chronic obstructive pulmonary disease andasthma. Sanders et al. disclose methods for treating the autonomicnervous system; for example, treating autonomic nervous system disorderssuch as excessive sweating, excessive salivation, asthma, etc., usingnaturally existing botulinum toxins. The disclosure of Sander et al. isincorporated in its entirety by reference herein. In one embodiment,substantially similar methods to that of Sanders et al. can be employed,but using a modified neurotoxin, to treat autonomic nervous systemdisorders such as the ones discussed above. For example, a modifiedneurotoxin can be locally applied to the nasal cavity of the mammal inan amount sufficient to degenerate cholinergic neurons of the autonomicnervous system that control the mucous secretion in the nasal cavity.

Pain that can be treated by a modified neurotoxin includes pain causedby muscle tension, or spasm, or pain that is not associated with musclespasm. For example, Binder in U.S. Pat. No. 5,714,468 discloses thatheadache caused by vascular disturbances, muscular tension, neuralgiaand neuropathy can be treated with a naturally occurring botulinumtoxin, for example Botulinum type A. The disclosures of Binder areincorporated in its entirety herein by reference. In one embodiment,substantially similar methods to that of Binder can be employed, butusing a modified neurotoxin, to treat headache, especially the onescaused by vascular disturbances, muscular tension, neuralgia andneuropathy. Pain caused by muscle spasm can also be treated by anadministration of a modified neurotoxin. For example, a botulinum type Efused with a leucine-based motif, preferably at the carboxyl terminal ofthe botulinum type E light chain, can be administered intramuscularly atthe pain/spasm location to alleviate pain.

Furthermore, a modified neurotoxin can be administered to a mammal totreat pain that is not associated with a muscular disorder, such asspasm. In one broad embodiment, methods of the present invention totreat non-spasm related pain include central administration orperipheral administration of the modified neurotoxin.

For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses that abotulinum toxin conjugated with a targeting moiety can be administeredcentrally (intrathecally) to alleviate pain. The disclosures of Fosteret al. are incorporated in its entirety by reference herein. In oneembodiment, substantially similar methods to that of Foster et al. canbe employed, but using the compositions described herein to treat pain.The pain to be treated can be an acute pain or chronic pain.

An acute or chronic pain that is not associated with a muscle spasm canalso be alleviated with a local, peripheral administration of themodified neurotoxin to an actual or a perceived pain location on themammal. In one embodiment, the modified neurotoxin is administeredsubcutaneously at or near the location of pain, for example, at or neara cut. In some embodiments, the modified neurotoxin is administeredintramuscularly at or near the location of pain, for example, at or neara bruise location on the mammal. In some embodiments, the modifiedneurotoxin is injected directly into a joint of a mammal, for treatingor alleviating pain caused by arthritic conditions. Also, frequentrepeated injection or infusion of the modified neurotoxin to aperipheral pain location is within the scope of the present invention

Routes of administration for such methods are known in the art andeasily adapted to the methods described herein by the skilledpractitioner (e.g., see for example, Harrison's Principles of InternalMedicine (1998), edited by Anthony Fauci et al., 14.sup.th edition,published by McGraw Hill). By way of non-limiting example, the treatmentof a neuromuscular disorder can comprise a step of locally administeringan effective amount of the molecule to a muscle or a group of muscles,the treatment of an autonomic disorder can comprise a step of locallyadministering an effective of the molecule to a gland or glands, and thetreatment of pain can comprise a step of administering an effectiveamount of the molecule the site of the pain. In addition, the treatmentof pain can comprise a step of administering an effective amount of amodified neurotoxin to the spinal cord.

The embodiments described here and in the following examples are forillustrative purposes only, and various modifications or changesapparent to those skilled in the art are included within the scope ofthe invention.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to describe the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A botulinum neurotoxin (BoNT) polypeptide comprising:    -   a) a protease domain;    -   b) a protease cleavage site;    -   c) a translocation domain; and    -   d) a modified receptor binding domain of Clostridial botulinum        serotype B (B-H_(c)), comprising one or more substitution        mutations corresponding to substitution mutations in serotype B,        strain 1, selected from the group consisting of:        -   V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y;            S1199F; S1199L; S1201V; and combinations thereof.-   2. The BoNT polypeptide of paragraph 1, wherein the modified    (B-H_(c)) comprises two substitution mutations.-   3. The BoNT polypeptide of paragraph 2, wherein the two substitution    mutations correspond to E1191M and S1199L, E1191M and S1199Y, E1191M    and S1199F, E1191Q and S1199L, E1191Q and S1199Y, or E1191Q and    S1199F.-   4. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191M and S1199L.-   5. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191M and S1199Y.-   6. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191M and S1199F.-   7. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191Q and S1199L.-   8. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191Q and S1199Y.-   9. The BoNT polypeptide of one of paragraphs 2-3, wherein the two    substitution mutations correspond to E1191Q and S1199F.-   10. A botulinum neurotoxin (BoNT) polypeptide comprising:    -   a) a protease domain;    -   b) a protease cleavage site;    -   c) a translocation domain; and    -   d) a modified receptor binding domain of Clostridial botulinum        serotype B (B-H_(c)), comprising a substitution mutation at a        position corresponding to S1199 or S1201 of serotype B, strain        1.-   11. The BoNT polypeptide of paragraph 10 wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or reduced binding of the modified B-H_(c) to human Syt I    as compared to an identical molecule lacking the substitution    mutation.-   12. The BoNT polypeptide of paragraph 10 wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or increased binding of the modified B-H_(c) to human Syt    I as compared to an identical molecule lacking the substitution    mutation.-   13. The BoNt polypeptide of any one of paragraphs 11-12 wherein the    substitution mutation is selected from the group consisting of A, R,    N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y and V substituted for    S.-   14. The BoNt polypeptide of any one of paragraphs 11-13 wherein the    substitution mutation is a non-naturally occurring amino acid    substituted for S.-   15. The BoNT polypeptide of any one of paragraphs 1-14, wherein the    modified B-H_(c) is of strain 1.-   16. The BoNT polypeptide of any one of paragraphs 1-15 wherein the    protease domain, translocation domain, and protease cleavage site    are from serotype selected from the group consisting of A, B, C, D,    E, F, G, and combinations thereof.-   17. The BoNT polypeptide of paragraph 16, wherein the protease    domain, translocation domain, and protease cleavage site are from    serotype B, strain 1.-   18. The BoNT polypeptide of paragraph 16, wherein the protease    domain, translocation domain, and protease cleavage site are from    serotype A, strain 1.-   19. A polypeptide comprising a modified receptor binding domain of    Clostridial botulinum serotype B (B-H_(c)) comprising one or more    substitution mutations corresponding to substitution mutations in    serotype B, strain 1, selected from the group consisting of V1118M;    Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L;    S1201V; and combinations thereof.-   20. The polypeptide of paragraph 19, wherein the modified (B-H_(c))    comprises two substitution mutations.-   21. The polypeptide of paragraph 20, wherein the two substitution    mutations correspond to E1191M and S1199L, E1191M and S1199Y, E1191M    and S1199F, E1191Q and S1199L, E1191Q and S1199Y, or E1191Q and    S1199F.-   22. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191M and S1199L.-   23. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191M and S1199Y.-   24. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191M and S1199F.-   25. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191Q and S1199L.-   26. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191Q and S1199Y.-   27. The polypeptide of one of paragraphs 20-21, wherein the two    substitution mutations correspond to E1191Q and S1199F.-   28. A polypeptide comprising a modified receptor binding domain of    Clostridial botulinum serotype B (B-H_(c)) comprising a substitution    mutation at a position corresponding to S1199 or S1201 of serotype    B, strain 1.-   29. The polypeptide of paragraph 28, wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or reduced binding of the modified B-H_(c) to human Syt I    as compared to an identical molecule lacking the substitution    mutation.-   30. The polypeptide of paragraph 28, wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or increased binding of the modified B-H_(c) to human Syt    I as compared to an identical molecule lacking the substitution    mutation.-   31. The polypeptide of any one of paragraphs 29-30 wherein the    substitution mutation is selected from the group consisting of A, R,    N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y and V substituted for    S.-   32. The polypeptide of any one of paragraphs 29-31 wherein the    substitution mutation is a non-naturally occurring amino acid    substituted for S.-   33. The polypeptide of any one of paragraphs 19-32, wherein the    modified B-H_(c) is of strain 1.-   34. A chimeric molecule comprising a first portion that is a    modified receptor binding domain of Clostridial botulinum serotype B    (B-H_(e)) linked to a second portion, wherein the modified B-H_(c)    comprises one or more substitution mutations corresponding to    substitution mutations in serotype B, strain 1, selected from the    group consisting of:    -   V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F;        S1199L; S1201V and combinations thereof.-   35. The chimeric molecule of paragraph 33, wherein the modified    B-H_(c) comprises two substitution mutations.-   36. The chimeric molecule of paragraph 35, wherein the two    substitution mutations correspond to E1191M and S1199L, E1191M and    S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, or    E1191Q and S1199F.-   37. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191M and S1199L.-   38. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191M and S1199Y.-   39. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191M and S1199F.-   40. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191Q and S1199L.-   41. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191Q and S1199Y.-   42. The chimeric molecule of one of paragraphs 35-36, wherein the    two substitution mutations correspond to E1191Q and S1199F.-   43. The chimeric molecule of paragraph 34, wherein the modified    B-H_(c) comprises a modified receptor binding domain of Clostridial    botulinum serotype B (B-H_(c)) comprising a substitution mutation at    a position corresponding to S1199 or S1201 of serotype B, strain 1.-   44. The chimeric molecule of paragraph 43, wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or reduced binding of the modified B-H_(c) to human Syt I    as compared to an identical molecule lacking the substitution    mutation.-   45. The chimeric molecule of paragraph 43, wherein the substitution    mutation produces enhanced binding of the modified B-H_(c) to human    SytII and/or increased binding of the modified B-H_(c) to human Syt    I as compared to an identical molecule lacking the substitution    mutation.-   46. The chimeric molecule of any one of paragraphs 44-45 wherein the    substitution mutation is selected from the group consisting of A, R,    N, D, C, Q, E, G, H, I, L, K, M, F, P, T, W, Y and V substituted for    S.-   47. The chimeric molecule of any one of paragraphs 44-46 wherein the    substitution mutation is a non-naturally occurring amino acid    substituted for S.-   48. The chimeric molecule of any one of paragraphs 43-47, wherein    the modified B-H_(c) is of strain 1.-   49. The chimeric molecule of any one of paragraphs 32-48, wherein    the first portion and the second portion are linked covalently.-   50. The chimeric molecule of any one of paragraphs 32-48, wherein    the first portion and the second portion are linked non-covalently.-   51. The chimeric molecule of any one of paragraphs 32-50 wherein the    second portion is selected from the group consisting of a small    molecule, a nucleic acid, a short polypeptide and a protein.-   52. The chimeric molecule of paragraph 51, wherein the second    portion is a bioactive molecule.-   53. The chimeric molecule of paragraph 51 or 52, wherein the second    portion is a therapeutic polypeptide or non-polypeptide drug.-   54. A nucleic acid comprising a nucleotide sequence that encodes the    polypeptide or chimeric molecule of any one of paragraphs 1-53.-   55. A nucleic acid vector comprising the nucleic acid of paragraph    54.-   56. A cell comprising the nucleic acid vector of paragraph 55 or the    nucleic acid of paragraph 54.-   57. A cell expressing the polypeptide or chimeric molecule of any    one of paragraphs 1-53.-   58. A pharmaceutical composition comprising the botulinum neurotoxin    (BoNT) polypeptide of any one of paragraphs 1-18, or the chimeric    molecule of any one of paragraphs 34-53, or the nucleic acid vector    of paragraph 55 or the nucleic acid of paragraph 54.-   59. The pharmaceutical composition of paragraph 58, further    comprising a pharmaceutically acceptable excipient.-   60. A kit comprising a pharmaceutical composition of paragraph 58 or    59 and directions for therapeutic administration of the    pharmaceutical composition.-   61. A method to produce a botulinum neurotoxin (BoNT) polypeptide,    the method comprising the steps of culturing the host cell of    paragraph 57 under conditions wherein said BoNT polypeptide is    produced.-   62. The method of paragraph 61 further comprising recovering the    BoNT polypeptide from the culture.-   63. A method for treating a condition associated with unwanted    neuronal activity comprising administering a therapeutically    effective amount of the BoNT polypeptide of any one of paragraphs    1-18 to a subject to thereby contact one or more neurons exhibiting    unwanted neuronal activity, to thereby treat the condition.-   64. The method of paragraph 63, wherein the condition is selected    from the group consisting of, spasmodic dysphonia, spasmodic    torticollis, laryngeal dystonia, oromandibular dysphonia, lingual    dystonia, cervical dystonia, focal hand dystonia, blepharospasm,    strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal    spasticity and other voice disorders, spasmodic colitis, neurogenic    bladder, anismus, limb spasticity, tics, tremors, bruxism, anal    fissure, achalasia, dysphagia and other muscle tone disorders and    other disorders characterized by involuntary movements of muscle    groups, lacrimation, hyperhydrosis, excessive salivation, excessive    gastrointestinal secretions, secretory disorders, pain from muscle    spasms, headache pain, and dermatological or aesthetic/cosmetic    conditions.-   65. The botulinum neurotoxin (BoNT) polypeptide of any one of    paragraphs 1-18, the pharmaceutical composition of paragraph 58 or    59, or the chimeric molecule of any one of paragraphs 34-53, or the    polypeptide of any one of paragraphs 19-33, for use in medicine.-   66. The botulinum neurotoxin (BoNT) polypeptide of any one of    paragraphs 1-18, the pharmaceutical composition of paragraph 58 or    59, or the chimeric molecule of any one of paragraphs 34-53, or the    polypeptide of any one of paragraphs 19-33, for use in treating a    condition associated with unwanted neuronal activity.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES

The following experiments were performed to determine if it is possibleto change the binding affinity of BoNT/B to human Syt II by modifyingthe BoNT/B receptor binding domain. The hypothesis is based on a seriesof previous studies: (1) It has been shown in 1998 that a naturallyoccurring BoNT/B subtype toxin, BoNT/B2, exhibits ˜4 fold lower bindingaffinity to Syt II than BoNT/B ²⁸(also defined as BoNT/B1, FIG. 2F).This affinity difference was demonstrated to be due to a few amino aciddifferences within their receptor binding domains in 2003 ²⁹(FIG. 2F,G), demonstrating for the first time that changing residues within thereceptor binding domain of BoNT/B can change the binding affinity to SytII. These studies also identified key residues that influence bindingaffinity to Syt II (FIG. 2G). (2) It has been reported in 2004 thatsingle residue mutations within the receptor binding domain of BoNT/Aand BoNT/B can dramatically change the toxicity and potency of thesetoxins (FIG. 2H), demonstrating that changes in receptor-bindingaffinity can translate into changes of toxicity and potency of toxins³⁰. (3) The co-crystal structure of BoNT/B bound to rat Syt II has beensolved ^(31,32), and key residues that form the binding site for Syt IIhave been resolved^(31,32). These previous studies all utilized therodent Syt II, but not human Syt II.

Target residues for engineering BoNT/B receptor binding domain to changeits binding affinity to human Syt II were identified from all theseprevious studies with rodent Syt II binding.

The receptor binding domain of BoNT/B is well defined ¹. Previousstudies established that changing residues within the receptor bindingdomain of BoNT/B can modulate the binding affinity of BoNT/B to rat ormouse Syt II ^(29,30). Co-crystal structure of BoNT/B bound to rat SytII has also been solved by two studies in 2006 ^(31,32). The residuechange in human Syt II is a relatively conservative change from F to L,both are hydrophobic residues. However, the difference in the bindingaffinity of BoNT/B for rodent Syt II is significantly higher than forhuman Syt II. Furthermore, it is not obvious how the binding interactionbetween BoNT/B and human Syt II might be modified to compensate for thelack of this phenylalanine residue in the middle of the binding site.Whereas positive binding interactions can be envisaged (and visualizedin published crystal structures) between WT BoNT/B-H_(C) and rat ormouse Syt II, e.g. involving stacking or packing of hydrophobic rings,or between a WT BoNT/B-H_(C) and a modified human Syt II in which thephenylalanine is substituted into the sequence; such interactions maynot be reproducible between a modified BoNT/B-HC and a WT human Syt IIprotein. This suggests that changing a few or even one residue in BoNT/Bmight not be able to restore/improve binding to human Syt II withoutmajor changes in the global structure of BoNT/B-Syt II complexes.

The conserved phenylalanine at position 54 forms multiple hydrophobiccontacts with BoNT/B. Because leucine (in humans) is also hydrophobic,disruption of BoNT/B binding is likely due to size/shape differencesbetween phenylalanine and leucine. The key to the invention wastherefore to identify possible changes in BoNT/B-H_(C) region that mayaccommodate and compensate for the change from phenylalanine to leucine.The approach was two-fold: to focus on residues directly contactingphenylalanine 54 in rodent Syt II: or to focus on residues within thesurrounding region of BoNT.B-Hc, which might compensate for the lack ofa positive binding interaction with phenylalanine at position 54. Theseresidues that are potentially within the corresponding binding regionbetween BoNT/B and human Syt II were judged by reference to theBoNT/B-rat Syt II co-crystal structure (FIG. 21), to possibly includeY1181, P1197, A1196, F1204, F1194, P1117, W1178, Y1183, V1118, S1116,K1113, K1192, S1199, S1201, E1191, E1245, and Y1256. Residues 1117,1191, and 1199 have also been shown to be among the list of residuesthat influence binding of BoNT/B2 to rodent Syt II in an earlier study(FIG. 2G)²⁹. Because the precise effect from residue substitutions isimpossible to predict, a “trial-and-error” approach was employed. Atfirst, single residue substitutions were carried out, followed byselected combinations. Specifically, each of the listed key residueswere systematically substituted with hydrophobic residues with differentsizes—with the screen limited to hydrophobic residues in order to ensurethat important hydrophobic contacts were maintained. These hydrophobicsubstitution residues include: V, I, L, M, F, W, C, and other lesshydrophobic amino acids including A, Y, H, T, S, P, Q, N, and G.

A key to the success of the invention was to develop a feasible andeconomical way for screening mutants. The basic approach was to detectbinding of soluble recombinant BoNT/B-H_(C) to immobilized mouseSyt II(F54L) in pull-down assays as described in FIG. 2C. However, it was notfeasible to purify all mutants for pull-down assays. Therefore, whetherit was possible to pull down BoNT/B-H_(C) from a small amount ofbacterial lysates directly with Syt II, without the need forpurification, was tested. The rationale was that the binding affinity ofBoNT/B-Syt II might be high enough for this approach (Kd˜0.23 nM) ²⁰.Indeed, it was found that immobilized rat Syt II could “affinity-purify”enough WT BoNT/B-H_(C) directly from merely 6 ml of bacterial lysates(FIG. 3A). This newly developed method greatly simplified the effort toscreen a fairly large number of BoNT/B-H_(C) mutants. Using this method,screening of BoNT/B-H_(C) mutants for their binding to both a mouse SytII 1-87 (m-Syt II) and a mutated mouse Syt II that mimicking human SytII sequence (F54L, h-Syt II) was tested. Bound materials were subjectedto immunoblot analysis detecting BoNT/B-H_(C) using the anti-HA antibody(FIG. 3A).

The majority of mutants were found to fall into two categories: (1) failto bind m-Syt II and h-Syt II, such as F1204L and V1118W (FIG. 3B); (2)still bind m-Syt II, but fail to bind h-Syt II, such as F1204W andE1191W (FIG. 3B). These binding results are largely omitted here excepta few examples illustrated in FIG. 3A.

Among mutants screened, a few that bound both m-Syt II and h-Syt II,including V1118M, S1199Y/L/F, Y1183M, S1201V, E1191M/I/Q/T (FIG. 3B)were identified. Thus, these residues were determined to be at keypositions for accommodating the L residues in human Syt II or forcompensating for the lack of phenylalanine residue at this position inhuman Syt II. Although human Syt I is expressed at significantly lowerlevels in motor neurons than human Syt II, it is nevertheless animportant and capable toxin receptor, as demonstrated by theeffectiveness of BoNT/B in patients. In order to achieve the highestpossible binding to human neurons, in some aspects the modified BoNT/Bmutants should desireably not adversely affect binding to human Syt I.Ideally, they may even increase binding to Syt I. Therefore, the bindingof selected BoNT/B mutants to immobilized human Syt I, using the samesmall-scale pull-down assay (FIG. 4A) was further examined. Because SytI binding to BoNT/B has a lower affinity as compared to Syt II, itrequires the presence of lipid co-receptor gangliosides ^(10,20). Thisneed was addressed by adding purified brain gangliosides into bacteriallysates in the pull-down assays. As indicated in FIG. 4A, human Syt Ifragment (1-80) containing the toxin binding site was purified asGST-tagged proteins and immobilized on beads to pull down WT and mutantBoNT/B-HC, with and without the presence of gangliosides (Gangl). Asexpected, WT BoNT/B-HC binds Syt I only in the presence of gangliosides.It was found that the mutants E1191M and E1191Q significantly increasedbinding to Syt I: these mutants can even bind to human Syt I withoutgangliosides (FIG. 4A). Other mutants either reduced binding to Syt I(e.g. V118M) or maintained the similar levels of binding as compared toWT BoNT/B-H_(C) (e.g. S1201V). This indicates that E1191M and E1191Q aremutants that both enable binding to human Syt II and enhance binding tohuman Syt I.

Mutation V1118M was also of interest as it binds to human Syt II, butnot human Syt I. Therefore, it has the potential to be used to createtherapeutic toxins that are more specific for neurons that express SytII than the WT BoNT/B in humans, thus reducing non-specific entry intoSyt-I expressing cells in humans.

Using E1191M as an example, its interactions with human Syt II werefurther validated using purified recombinant proteins, which allows usto compare binding of equal amounts of WT BoNT/B-H_(C) and the E1191Mmutant to m-Syt II and h-Syt II (FIG. 4B). E1191M was found to bind toboth m-Syt II and h-Syt II without gangliosides, and adding gangliosidesfurther elevated the binding (FIG. 4B). These results confirmed thatE1191M gains the ability to bind human Syt II in the absence ofgangliosides and can form high-affinity complexes with human Syt II inthe presence of the lipid co-receptor gangliosides.

Using E1191M/Q as the backbone, experiments were performed to analyzewhether combining it with other residue substitutions may furtherenhance binding to human Syt I/II. Combining S1199L/Y/ or /F withE1191M/or Q generated double mutants that display significantly higherbinding to human Syt II (FIG. 5A). For instance, E1191M/S1199Y achievedsimilar levels of binding to both m-Syt II and h-Syt II (FIG. 5A, lane 5and 6). This was a significant enhancement as compared to E1191M alone,which mediated less binding to h-Syt II than its binding to m-Syt II(FIG. 4B). Furthermore, all selected double mutants displayedsignificantly higher binding to human Syt I than WT BoNT/B-H_(C) (FIG.5B).

Using E1191M/S1199Y as an example, binding of WT, E1191M, andE1191M/S1199Y to h-Syt II were further compared using equal amounts ofpurified recombinant proteins. As shown in FIG. 6A, WT BoNT/B-H_(C)could not bind to h-Syt II in the absence of gangliosides under thecurrent assay conditions. E1191M showed a modest binding to h-Syt IIwithout gangliosides, while binding of E1191M/S1199Y to h-Syt II wassignificantly enhanced as compared to E1191M alone, especially withoutgangliosides (comparing lanes 6 versus 8). Furthermore, both E1191M andE1191M/S1199Y significantly enhanced binding to human-Syt I as comparedto WT BoNT/B-H_(C) (FIG. 6B).

Binding of WT BoNT/B-H_(C) to m-Syt II is known to have a high affinity^(20,21). Thus the binding between E1191M/S1199Y to h-Syt II versus the“golden standard”: WT BoNT/B-HC binding to m-Syt II was compared. Asshown in FIG. 6C, titration of BoNT/B-H_(C) concentrations revealed thatE1191M/S1199Y has similar levels of binding at all concentrations as WTbinding to m-Syt II. The Kd was estimated to be ˜19 nM betweenE1191M/S1199Y and h-Syt II, and ˜68 nM for WT BoNT/B-H_(C) binding tom-Syt II under this assay condition (FIG. 6D) This is a giganticimprovement for binding h-_(s)yt II as compared to WT BoNT/B-H_(C),which failed to bind h-Syt II in these assay conditions (FIG. 6A). Inconclusion, combining E1191M with S1199Y provided a synergisticimprovement in binding affinity, outweighing an additive improvementover the E1191M mutant and yielded new BoNT/B-H_(C) mutants with highaffinity binding to both human Syt I and Syt II. By contrast,combinations of some other beneficial individual mutations did notresult in further improved double-mutant BoNT/B-H_(C) domains.

Finally, whether E1191M/S1199Y mutant can recover the binding to h-SytII on the neuron surface was examined. Cultured rat hippocampal neuronsonly express Syt I, but not Syt II. Syt I was knocked down (KD) in theseneurosn and then replaced with exogenous m-Syt II, m-Syt II (F54L), andh-Syt II via lentiviral transduction. Binding of WT BoNT/B-H_(C) andE1191M/S1199Y to these neurons was then tested (FIG. 7). WT BoNT/B-H_(C)only bound to m-Syt II, whereas E1191M/S1199Y bound to both m-Syt II(F54L) and h-Syt II on the neuron surface, demonstrating thatE1191M/S1199Y mutant can use h-Syt II as a functional receptor inneurons.

Materials and Methods

Antibodies and materials: The mouse monoclonal anti-HA antibody waspurchased from Covance (16B12). Bovine mixed brain gangliosides werepurchased from Matreya LLC (Pleasant Gap, Pa.) and were reconstituted inTris-buffered saline (TBS: 20 mM Tris, 150 mM NaCl) as previouslydescribed ⁹. BoNT/B (Okra) was purified in E. Johnson's lab (Madison,Wis.) from indicated strains.

cDNA and constructs: DNA encoding BoNT/B-H_(C) (residue 856-1291, basedon GenBank access No:AB232927.1) was synthesized by Geneart Inc. and itscodon has been optimized for expression in E. Coli. DNA encodingBoNT/B-H_(C) was subcloned into pET28a vector, with both a His6 tag anda HA tag (YPYDVPDYA) fused to its N-terminus. Mutations in BoNT/B-H_(C)were generated via PCR using Quickchange Site-directed Mutagenesis Kit(Agilent Technologies, Calif.), following the manufacturer's manual. Thefollowing DNA were generously provided by indicated groups: rat Syt I(T.C. Sudhof, Palo Alto, Calif.), mouse Syt II (M. Fukuda, Ibaraki,Japan), human Syt I (R. B. Sutton, Lubbock, Tex.). GST tagged Syt I/IIfragments and Syt II mutations were described previously ^(10,13,14).All constructs were verified by sequencing.

Protein expression and purification: WT and mutants of BoNT/B-H_(C) wereexpressed as His6 tagged recombinant proteins in E. Coli. Syt I/IIfragments and mutants were expressed as GST tagged recombinant proteinsin E. Coli. Both GST-fusion and His₆-fusion proteins were purified aspreviously described ⁹, with the induction temperature at 20° C.overnight with 0.25 mM IPTG.

GST pull-down assays: Two types of pull-down assays were carried out.The first series were used to screen binding of mutant BoNT/B-H_(C) toGST-tagged mouse Syt II (m-Syt II) and a mutant mouse Syt II (F54L) thatmimicking human Syt II sequence (designated as h-Syt II in Examples 1 to6). Briefly, 6 ml of E. Coli expressing BoNT/B-H_(C) were spin down,re-suspended in 800 μl TBS, sonicated, and then incubated with 2% TritonX-100 for 1 hr at 4° C. Samples were then spun down at maximal speed for15 min in a microcentrifuge at 4° C. The supernatants were collected andwere used for pull-down assays by incubating with 10 μg of Syt proteinsimmobilized on glutathione-Sepharose beads (GE bioscience, Piscataway,N.J.) at 4° C. for 1 hr. Samples were washed three times in washingbuffer (TBS+0.5% Triton), and analyzed by immunoblot assays detectingBoNT/B-H_(C) using the anti-HA antibody. For mutants with enhancedbinding to h-Syt II, further pull-down assays were carried out bypurifying these mutant BoNT/B-H_(C) as His6 tagged proteins as describedpreviously ⁹. Pull-down assays were then carried out using immobilizedSyt fragments in 100 μl TBS buffer plus 0.5% Triton X-100, with orwithout gangliosides (60 μg/ml), for 1 hr at 4° C. Beads were washedthree times using TBS buffer plus 0.5% Triton X-100. Ten percent ofbound materials were subjected to SDS-PAGE followed by immunoblotanalysis.

Immunostaining: Culture neurons were fixed with 4% paraformaldehyde,permeabilized with 0.25% Triton X-100, and subjected to immunostaininganalysis detecting both BoNT/B-H_(C) (with an HA antibody) and synapsin.Images were collected using a confocal microscope (Leica TCS SP5; 40×oilobjective).

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1. A botulinum neurotoxin (BoNT) polypeptide comprising: a) a proteasedomain; b) a protease cleavage site; c) a translocation domain; and d) amodified receptor binding domain of Clostridial botulinum serotype B(B-H_(c)), comprising one or more substitution mutations correspondingto substitution mutations in serotype B, strain 1, selected from thegroup consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T;S1199Y; S1199F; S1199L; S1201V; and combinations thereof. 2-66.(canceled)